High-Throughput Screening for Bacterial Glycosyltransferase Inhibitors.

Samir El Qaidi,Pradip Kumar Maity,Peter Mcdonald,Philip R Hardwidge,Digamber Rane,Chamani Perera,Anuradha Roy,Congrui Zhu

doi:10.3389/fcimb.2018.00435

Abstract

The enteropathogenic and enterohemorrhagic Escherichia coli NleB proteins as well as the Salmonella enterica SseK proteins are type III secretion system effectors that function as glycosyltransferase enzymes to post-translationally modify host substrates on arginine residues. This modification is unusual because it occurs on the guanidinium groups of arginines, which are poor nucleophiles, and is distinct from the activity of the mammalian O-linked N-acetylglucosaminyltransferase. We conducted high-throughput screening assays to identify small molecules that inhibit NleB/SseK activity. Two compounds, 100066N and 102644N, both significantly inhibited NleB1, SseK1, and SseK2 activities. Addition of these compounds to cultured mammalian cells was sufficient to inhibit NleB1 glycosylation of the tumor necrosis factor receptor type 1-associated DEATH domain protein. These compounds were also capable of inhibiting Salmonella enterica strain ATCC 14028 replication in mouse macrophage-like cells. Neither inhibitor was significantly toxic to mammalian cells, nor showed in vitro cross-reactivity with the mammalian O-linked N-acetylglucosaminyltransferase. These compounds or derivatives generated from medicinal chemistry refinements may have utility as a potential alternative therapeutic strategy to antibiotics or as reagents to further the study of bacterial glycosyltransferases.

Highlights

Many Gram-negative bacterial pathogens interact with mammalian cells by using a specialized type III secretion system (T3SS) to inject proteins directly into infected host cells (Notti and Stebbins, 2016; Deng et al, 2017)
Several death domaincontaining proteins have been described as substrates of some of the NleB/SseK orthologs, including the Fas-associated protein with death domain (FADD), tumor necrosis factor receptor type 1-associated DEATH domain protein (TRADD), and the receptor-interacting serine/threonine-protein kinase 1 (RIPK1) (Li et al, 2013)
We developed and optimized a high-throughput screening (HTS) assay for NleB1 inhibitors

Summary

INTRODUCTION

Many Gram-negative bacterial pathogens interact with mammalian cells by using a specialized type III secretion system (T3SS) to inject proteins directly into infected host cells (Notti and Stebbins, 2016; Deng et al, 2017). NleB1 Inhibitors protein substrates on arginine residues (Li et al, 2013; Pearson et al, 2013) This modification is unusual because it occurs on the guanidinium groups of arginines, which are poor nucleophiles. This modification is biologically important because the glycosylation of arginines on protein substrates disrupts the normal functioning of the innate immune system (Li et al, 2013; Pearson et al, 2013; El Qaidi et al, 2017). Antibiotic therapy against EHEC is typically contraindicated, because antibiotics may induce increased Shigalike toxin expression, exacerbating patient symptoms (Wong et al, 2000) This motivated our work, which sought to identify small molecule inhibitors of NleB1 as a potential alternative therapeutic strategy to antibiotics. Such compounds may be of utility as mechanistic probes for understanding the mechanism of the NleB/SseK effector family

RESULTS

DISCUSSION

EXPERIMENTAL PROCEDURES